Gene modified HEK293 cell as well as preparation method and application thereof
By genetically modifying HEK293 cells and preparing mIL-21 and 4-1BBL gene-expressing cells as trophoblast cells, combined with IL-2 and other cytokines, the efficient expansion and high-purity acquisition of NK cells were achieved, solving the existing technical problems The problem of slow expansion rate and insufficient lethality of NK cells in China.
Patent Information
- Application Number
- CN202411638892.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-18
- Publication Date
- 2025-01-21
AI Technical Summary
The prior art is difficult to efficiently obtain a large number of highly lethal NK cells. The traditional methods are costly, cumbersome to operate, long cycles and limited amplification.
By genetically modifying HEK293 cells, inserting the coding genes for mIL-21 and 4-1BBL, and through lentiviral packaging and irradiation treatment, HEK293 cells stably expressing these genes were prepared. These genetically modified HEK293 cells serve as trophoblast cells, mixed with NK cells, and utilize IL-2 and other cytokines for efficient in vitro expansion of NK cells.
The rapid expansion and high-purity acquisition of NK cells are achieved, and the killing power of NK cells is significantly enhanced, which solves the problems of slow expansion speed and insufficient killing power of NK cells in the existing technology.
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Figure CN119331825A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of gene-edited biological cells, and in particular to a gene-modified HEK293 cell and application of the cell in efficient amplification and culture of NK cells. Background Art
[0002] NK cells are a type of cell that can kill tumor cells with a broad spectrum, which is different from the T cell-dependent mechanism of the major histocompatibility complex (MHC)-restricted T cell receptor (TCR). Therefore, the use of allogeneic NK cells to treat cancer will not cause graft-versus-host disease (GVHD), which makes NK cells show great potential in cancer treatment.
[0003] However, the number and killing function of NK cells are directly related to the therapeutic effect. The problem in treatment is that it is very difficult to obtain a large number of NK cells.
[0004] At the current stage, the methods used to enrich and expand NK cells in peripheral blood are: first, most of them rely on the use of invasive blood cell separation, large magnetic separation equipment and cytokines. This method has high culture costs, cumbersome operations, long culture cycles, and a small number of cell expansion. Second, trophoblast cells are used to expand and stimulate NK cell proliferation, such as Wilms tumor cell lines, EBV-transformed lymphoblasts, and chronic myeloid leukemia cell lines K562 cells; but this NK cell expansion method has a relatively slow cell expansion rate, and the resulting NK cells have weak lethality. Summary of the invention
[0005] Based on the above problems, the problem to be solved by the present invention is to provide a gene-edited HEK293 cell, and a method for efficiently expanding and culturing NK cells using such a cell.
[0006] The present invention provides a genetically modified HEK293 cell, wherein the coding genes of mIL-21 and 4-1BBL are inserted into the genome of the cell; the amino acid sequences corresponding to the coding genes of mIL-21 and 4-1BBL are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.
[0007] The present invention also provides a method for preparing the HEK293 cells, comprising the following steps: Construction of lentiviral expression plasmid: constructing the coding gene fragments of mIL-21 and 4-1BBL on lentiviral plasmids respectively to obtain lentiviral expression plasmids; Lentivirus packaging: adding lentivirus expression plasmid and transfection reagent to 293T cell solution for lentivirus packaging, collecting the supernatant, filtering and concentrating to obtain lentivirus concentrate; Cultivation of gene-modified HEK293 cells: HEK293 cells were inoculated into the wells of a culture plate containing a culture medium, a lentivirus concentrate was added into the wells of the culture plate, and the cells were incubated in an incubator at 37° C. and 5% CO 2 . After cessation of the culture, HEK293 cells modified with the mIL-21 and 4-1BBL genes were obtained.
[0008] Preferably, the HEK293 cells modified with mIL-21 and 4-1BBL genes are further treated as follows: Irradiation treatment of HEK293 cells: After the mIL-21 and 4-1BBL-modified HEK293 cells were irradiated with 100 Gy of γ-rays for 0.5 h, irradiated HEK293 cells were obtained; the cells were transferred to liquid nitrogen for storage and used later.
[0009] In one embodiment, in the method for preparing HEK293 cells, the lentivirus packaging process further comprises the following steps: The lentiviral envelope plasmid pMD2.G, the lentiviral expression plasmid, the auxiliary plasmid pMDLg / pRRE, and the auxiliary plasmid pRSV-Rev were mixed to obtain a four-plasmid mixture; wherein the mass ratio of the lentiviral envelope plasmid pMD2.G, the lentiviral expression plasmid, the auxiliary plasmid pMDLg / pRRE, and the auxiliary plasmid pRSV-Rev was 1:4:3:2; Lipofectamine 3000 transfection agent was added to the four-plasmid mixture at a volume ratio of 1:2 and mixed evenly, and the mixed four-plasmids were transferred into 293T cells to obtain a lentiviral vector solution overexpressing mIL-21 and 4-1BBL; The lentiviral vector solution is centrifuged, the supernatant is collected, filtered and concentrated to obtain a lentiviral concentrate.
[0010] In one embodiment, in the method for preparing EK293 cells, the culturing process of genetically modified HEK293 cells comprises the following steps: The cell density per well was 1×10 5 HEK293 cells were seeded into the wells of the culture plate and each well was filled with DMEM medium containing 10% FBS and cultured in an incubator at 37°C and 5% CO2 for 24 h. After 24 h of culture, the culture medium waste liquid was discarded, and 300 μL of fresh serum-free DMEM medium was added to the wells of the culture plate, and the culture was continued for 1 h; After 1 hour of incubation, add the lentiviral concentrate to the wells of the culture plate according to MOI=5, and continue culturing for 6 hours; After 6 hours of incubation, add 700 μL of DMEM medium containing 10% FBS to the wells of the culture plate and continue to culture for 72 hours; After 72 h of culture, 1 mL of DMEM medium containing 1 ug / mL puromycin and 10% FBS was added to the culture plate and culture was continued for 24 h. After the culture was completed, HEK293 cells modified with mIL-21 and 4-1BBL genes were obtained.
[0011] The present invention also provides a method for amplifying and culturing NK cells using the HEK293 cells, comprising the following steps: On day 0, take 5 x 10 6 HEK293 cells treated with the above radiation and 2.5×10 6 PBMC or NK cells isolated from peripheral blood were added into culture bottles containing NK cell culture medium and cultured in an incubator at 37°C and 5% CO2. From the 3rd day to the 11th day, fresh NK cell culture medium is added to the culture flask every day; on the 7th day, the irradiated HEK293 cells are also added to the culture flask until the end of the 11th day of culture, and the cultured NK cells are collected.
[0012] Preferably, in the NK cell culture method, after obtaining the NK cells on the 11th day, the following treatment is further included: The collected NK cells were transferred to a centrifuge tube, centrifuged at 700 g for 10 min, and the supernatant was removed; the NK cell pellet was resuspended with freezing solution, and the obtained NK cell suspension was divided into freezing tubes, transferred to liquid nitrogen, and set aside.
[0013] In one embodiment, in the NK cell culture method, from day 3 to day 11, the amount of fresh NK cell culture medium added every day should maintain the cell density at 1.0×10 6 On the 7th day, the number of HEK293 cells supplemented with irradiation was 3×10 7 indivual.
[0014] In one embodiment, in the NK cell culture method, the NK cell culture medium is 581 culture medium, which contains heat-inactivated human AB serum with a final concentration of 5 v / v% and IL-2 with a final concentration of 200 U / mL.
[0015] The present invention utilizes mIL-21 and -1BBL encoding gene fragments to construct expression vector plasmids and package lentiviruses, and then infects HEK293 cells with lentiviruses to stably express each target protein in HEK293 cells; the irradiated genetically modified HEK293 cells are then used to lose their proliferation function, but retain their activity; the irradiated genetically modified HEK293 cells are used as trophoblast cells, mixed with PBMC or NK cells, and NK cells are amplified in vitro, and cytokine IL-2 is supplemented at the same time. Through the detection of NK cell proliferation, flow cytometry detection of NK purity, and the use of luciferase to detect lethality, the results show that the HEK293 cells overexpressing mIL-21 and 4-1BBL genes constructed by the present invention are stably transferred cells, which play the role of trophoblast cells, can make NK cells proliferate and grow rapidly, and the NK cells are of high purity and strong lethality. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 Flow cytometry of mIL-21 and 4-1BBL expressed on the surface of HEK293 cells in Example 2; wherein, Figure a is a flow cytometry of negative expression of 4-1BBL on the surface of blank HEK293 cells, Figure b is a flow cytometry of positive expression of 4-1BBL on the surface of 4-1BBL-mIL-21-HEK293 cells, Figure c is a flow cytometry of negative expression of mIL-21 on the surface of blank HEK293 cells, and Figure d is a flow cytometry of positive expression of mIL-21 on the surface of 4-1BBL-mIL-21-HEK293 cells; Figure 2 NK cell purity percentage curve diagram in Examples 4 and 6; Figure 3 This is a flow cytometry analysis of the CD3-CD56+ ratio of NK cells sampled on the 11th day of PBMC cell culture in Example 4; Figure 4 This is a flow cytometry analysis of the CD3-CD56+ ratio of NK cells sampled on the 11th day of PBMC cell culture in Example 6; Figure 5 The growth curve of the number of NK cells in Examples 3 to 6; Figure 6 The NK cell number expansion multiple curves in Examples 3 to 6; Figure 7 This is a curve diagram of the NK cell tumor killing rate in Example 4 and Example 6. DETAILED DESCRIPTION
[0017] The preferred embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings.
[0018] The process flow of the efficient expansion and cultivation of NK cells of the present invention is as follows: First, the coding gene fragments of mIL-21 and 4-1BBL were inserted into lentiviral expression plasmids respectively; Then, HEK293 cells are infected by transfection, and the coding genes of mIL-21 and 4-1BBL are inserted into the genome of HEK293 cells to obtain gene-modified HEK293 cells; Next, the genetically modified HEK293 cells were expanded and cultured, and then the HEK293 cells were irradiated; Finally, irradiated HEK293 cells were added to NK cell culture medium as trophoblast cells to achieve efficient in vitro expansion and culture of NK cells.
[0019] Specifically, the preparation process of HEK293 cells used in the efficient expansion and culture of NK cells includes the following steps: S11, designing or obtaining the coding gene fragments of mIL-21 and 4-1BBL whose amino acid sequences are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively; S12, inserting the coding gene fragments of mIL-21 and 4-1BBL into the lentiviral plasmid respectively to construct a lentiviral expression plasmid; S13, infecting 293T cells with the lentiviral expression plasmid to perform lentiviral packaging, collecting the supernatant, filtering and concentrating to obtain a lentiviral concentrate; S14, inoculating HEK293 cells into the wells of a culture plate containing NK cell culture medium, adding lentivirus concentrate into the wells of the culture plate, and incubating in an incubator at 37°C and 5% CO2 to obtain HEK293 cells modified with mIL-21 and 4-1BBL genes; S15. Irradiate the mIL-21 and 4-1BBL gene-modified HEK293 cells with 100 Gy of γ rays for 0.5 h to obtain irradiated HEK293 cells, which are then transferred to liquid nitrogen for storage and used later.
[0020] In step S11 of the above HEK293 cell preparation process, the amino acid sequences corresponding to the coding genes of mIL-21 and 4-1BBL are as follows: The amino acid sequence of mIL-21 is shown in SEQ ID NO.1: MALPVTALLLPLALLLHAARPHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQK HRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC; The amino acid sequence of 4-1BBL is shown in SEQ ID NO.2: MEYASDASLDPEAPWPPARARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYK EDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPPSPRSE.
[0021] The construction of the lentiviral expression plasmid in step S12 of the above HEK293 cell preparation process belongs to conventional technology and will not be described in detail.
[0022] In step S13 of the above HEK293 cell preparation process, the lentivirus packaging process also includes the following processing steps: S131, mixing the lentiviral envelope plasmid pMD2.G (Addgene, Plasmid #12259), the lentiviral expression plasmid, the auxiliary plasmid pMDLg / pRRE (Addgene Plasmid, #12251), the auxiliary plasmid pRSV-Rev (Addgene Plasmid, #12253), etc. at a mass ratio of 1:4:3:2 to obtain a four-plasmid mixture; S132, mixing the four plasmid mixture with Lipofectamine 3000 transfection agent at a volume ratio of 1:2, and adding the mixture to the 293T cell solution, and transferring the four plasmids into the 293T cells through the transfection agent to prepare a lentiviral vector solution overexpressing mIL-21 and 4-1BBL; S133, transfer the lentiviral vector solution into a centrifuge tube, centrifuge at 700g for 10 min, collect the supernatant, filter and concentrate the supernatant to obtain a lentiviral concentrate for later use.
[0023] Step S14 of the above HEK293 cell preparation process also includes the following processing steps: S141, according to the cell density of each well 1x10 5 HEK293 cells were inoculated into the wells of the culture plate, and each well was filled with 1 mL of DMEM medium containing 10% FBS, and cultured in an incubator at 37°C and 5% CO2 for 24 h; S142. After 24 h of culture, discard the culture medium, add 300 µL of fresh serum-free DMEM medium to the wells of the culture plate, and continue to incubate in a 37°C, 5% CO2 incubator for 1 h. S143. After 1 h of culture, add lentiviral concentrate to the wells of the culture plate according to MOI = 5, and continue to incubate in a 37°C, 5% CO2 incubator for 6 h; S144. After the 6-h incubation, add 700 µL of DMEM medium containing 10% FBS to the wells of the culture plate, and continue to incubate in a 37°C, 5% CO2 incubator for 72 h; After S145 and 72h of culture, 1 mL of DMEM medium containing 1 ug / mL puromycin and 10% FBS was added to the wells of the culture plate, and the cells were incubated in an incubator at 37°C and 5% CO2 for 24 h. After the culture was completed, HEK293 cells modified with mIL-21 and 4-1BBL genes were obtained. S146. Resuspend HEK293 cells in FBS freezing solution containing 10% DMSO, divide the resuspended solution into cryopreservation tubes, transfer to liquid nitrogen for freezing and set aside.
[0024] After obtaining the above HEK293 cells, the present invention also uses HEK293 cells as trophoblast cells to perform efficient expansion and culture of NK cells, and the process steps are as follows: S21, day 0, take 5×10 6 The irradiated HEK293 cells and 2.5×10 6 PBMC or NK cells isolated from peripheral blood were added to a T25 culture flask containing 5 mL of NK cell culture medium and cultured in an incubator at 37°C and 5% CO2. S22. From the 3rd day to the 11th day, NK cell culture medium is added to the culture flask every day; on the 7th day, irradiated HEK293 cells are also added to the culture flask until the 11th day of culture, and NK cells are collected; S23. The collected NK cells were transferred to a centrifuge tube, centrifuged at 700 g for 10 min, and the supernatant was removed; the NK cell pellet was resuspended with freezing solution, and the obtained NK cell suspension was divided into freezing tubes, transferred to liquid nitrogen for standby use; wherein the freezing solution was FBS freezing solution containing 10% DMSO.
[0025] In the above NK cell culture method, the NK cell culture medium is 581 culture medium, which contains heat-inactivated human AB serum with a final concentration of 5 v / v% and IL-2 with a final concentration of 200 U / mL.
[0026] In the NK cell expansion and culture process step S21, since PMBCs in peripheral blood contain 8% NK cells, PMBCs in peripheral blood can be used to culture NK cells.
[0027] In the NK cell expansion culture process step S22, from day 3 to day 11, the amount of NK cell culture medium added daily should maintain the cell density at 1.0×10 6 / mL; on the 7th day, 3×10 7 Irradiated HEK293 cells.
[0028] The present invention is further described in detail below through specific examples.
[0029] Example 1 mIL-21, 4-1BBL lentiviral packaging Mix 2 μg of lentiviral envelope plasmid pMD2.G (Addgene, Plasmid #12259), 8 μg of lentiviral expression plasmid, 6 μg of auxiliary plasmid pMDLg / pRRE (Addgene Plasmid, #12251), and 4 μg of auxiliary plasmid pRSV-Rev (Addgene Plasmid, #12253) to obtain a four-plasmid mixture; 1 mL of the four-plasmid mixture and 2 mL of Lipofectamine 3000 transfection agent were added to the 293T cell solution, and the four plasmids were transferred into the 293T cells through the transfection agent to prepare a lentiviral vector solution overexpressing mIL-21 and 4-1BBL; The lentiviral vector solution was centrifuged at 700 g for 10 min, the supernatant was collected, filtered and concentrated to obtain a lentiviral concentrate for later use.
[0030] Example 2 Cultivation and detection of HEK293 cells 2.1. According to the cell density of 1×10 per well 5 HEK293 cells were seeded in a 24-well culture plate, and 1 mL of 10% FBS DMEM medium was added to each well. The culture plate was moved to make the HEK293 cells evenly spread in the wells of the culture plate, and then transferred to a 37°C, 5v / v% CO2 incubator for 24h; 2.2 After 24 hours of culture, take out the culture plate from the incubator, discard the culture medium, add 300 µL serum-free DMEM culture medium to each well, and then put it back into the incubator for further incubation. Perform lentiviral transduction 1 hour later. 2.3. According to MOI=5, 50 μL of the lentiviral concentrate prepared in Example 1 was taken and added to each well of the culture plate in step 2.2, marked, and returned to the incubator for further incubation. This time was recorded as 0 h. 2.4. After incubation for 6 hours, take out the culture plate, add 700 μL of DMEM medium containing 10% FBS to each well, and return it to the incubator to continue culturing for 72 hours; 2.5. After 72 hours of culture, DMEM medium containing 1 ug / mL puromycin and 10% FBS was added to each well of the culture plate, and the culture plate was returned to the incubator for further culture for 24 hours. After the culture was completed, HEK293 cells stably expressing mIL-21 and 4-1BBL genes were obtained; 2.6. Irradiating the obtained HEK293 cells with 100 Gy gamma rays for 0.5 h to obtain irradiated HEK293 cells; 2.7. Resuspend HEK293 cells in FBS freezing solution containing 10% DMSO, divide the resuspended solution into cryopreservation tubes, transfer to liquid nitrogen for freezing and set aside.
[0031] After the culture in step 2.5 was completed, the expression of mIL-21 and 4-1BBL on the surface of HEK293 cells was analyzed by flow cytometry. Figure 1 As shown, Figure a is a flow cytometric graph showing negative expression of 4-1BBL on the surface of blank HEK293 cells, Figure b is a flow cytometric graph showing positive expression of 4-1BBL on the surface of 4-1BBL-mIL-21-HEK293 cells, Figure c is a flow cytometric graph showing negative expression of mIL-21 on the surface of blank HEK293 cells, and Figure d is a flow cytometric graph showing positive expression of mIL-21 on the surface of 4-1BBL-mIL-21-HEK293 cells.
[0032] Figure 1Figures b and d show that the positive rates of mIL-21 and 4-1BBL expressed on the surface of HEK293 cells were both above 98%, indicating that HEK293 cells modified with mIL-21 and 4-1BBL genes were successfully constructed.
[0033] Example 3 Expansion and culture of NK cells 3.1, take 2.5×10 6 NK cells and 5×10 6 The irradiated HEK293 cells obtained in step 2.6 of Example 2 were co-cultured in a T25 culture flask, the T25 culture flask contained 5 mL of 581 culture medium, the culture medium contained heat-inactivated human AB serum at a final concentration of 5 v / v% and IL-2 at a final concentration of 200 U / mL, and the T25 culture flask was placed in a 37° C., 5 v / v% CO2 incubator for culture; 3.2. From day 3 to day 11, fresh 581 medium was added every day, and the cell density was ensured to be 1.0×10 6 / mL until the end of the 11th day of culture; on the 7th day, 3.0×10 7 irradiated HEK293 cells obtained in step 2.6 of Example 2; 3.3. On the 11th day, collect NK cells and transfer them to a 250 mL centrifuge tube, and centrifuge at 700 g for 10 min. Remove the supernatant, resuspend the NK cell pellet with FBS freezing solution containing 10% DMSO, divide the NK cell suspension into target doses, quickly place it in a -80 °C refrigerator for 24 h, and then transfer it to liquid nitrogen for freezing and standby use.
[0034] Example 4 Proliferation and culture of PBMC cells 4.1, take 2.5×10 6 PBMC cells and 5.0×10 6 The irradiated HEK293 cells obtained in step 2.6 of Example 2 were co-cultured in a T25 culture flask, the T25 culture flask contained 5 mL of 581 culture medium, the culture medium contained a final concentration of 5 v / v% heat-inactivated human AB serum and a final concentration of 200 U / mL IL-2, and the T25 culture flask was placed in a 37° C., 5 v / v% CO2 incubator for culture; 4.2. From the 3rd day to the 11th day, fresh 581 medium was added every day to ensure that the cell density was 1.0×10 6 / mL until the end of the 11th day of culture; on the 7th day, 3.0×10 7 irradiated HEK293 cells obtained in step 2.6 of Example 2; 4.3. On day 11, collect the cells and transfer them to a 250 mL centrifuge tube, centrifuge at 700 g for 10 min, remove the supernatant, resuspend the NK cell pellet with FBS freezing solution containing 10% DMSO, divide the NK cell suspension into target doses, quickly place in a -80 °C refrigerator for 24 h, and then transfer to liquid nitrogen for cryopreservation for later use.
[0035] Example 5 (Comparative Example 1) Expansion of NK cells 5.1. According to the process steps of Example 2, irradiated K562 cells overexpressing mIL-21 and 4-1BBL genes were prepared and used for later use; 5.2, take 2.5×10 6 NK cells and 5.0×10 6 The irradiated K562 cells obtained in step 5.1 were co-cultured in a T25 culture flask, the T25 culture flask contained 5 mL of 581 culture medium, the culture medium contained heat-inactivated human AB serum at a final concentration of 5 v / v% and IL-2 at a final concentration of 200 U / mL, and the T25 culture flask was placed in a 37° C., 5 v / v% CO2 incubator for culture; 5.3. From day 3 to day 11, fresh DMEM medium was added every day to ensure that the cell density was 1.0×10 6 cells / mL until the end of the 11th day of culture; on the 7th day, 3.0×10 7 The irradiated K562 cells were prepared in step 5.1; 5.4. On day 11, collect the cells and transfer them to a 250 mL centrifuge tube, centrifuge at 700 g for 10 min, remove the supernatant, resuspend the NK cell pellet with FBS freezing solution containing 10% DMSO, divide the NK cell suspension into target doses, quickly place in a -80 °C refrigerator for 24 h, and then transfer to liquid nitrogen for cryopreservation for later use.
[0036] Example 6 (Comparative Example 2) Proliferation and culture of PBMC cells 6.1, take 2.5×10 6 PBMC cells and 5.0×10 6 The irradiated K562 cells obtained in step 5.1 of Example 5 were co-cultured in a T25 culture flask, the T25 culture flask contained 5 mL of DMEM culture medium, the culture medium contained heat-inactivated human AB serum at a final concentration of 5 v / v% and IL-2 at a final concentration of 200 U / mL, and the T25 culture flask was placed in a 37° C., 5 v / v% CO2 incubator for culture; 6.2. From day 3 to day 11, fresh DMEM medium was added every day to ensure that the cell density after daily replenishment volume was 1.0×10 6cells / mL until the end of the 11th day of culture; on the 7th day, 3.0×10 7 The irradiated K562 cells prepared in step 5.1 of Example 5; 6.3. On day 11, collect the cells and transfer them to a 250 mL centrifuge tube, centrifuge at 700 g for 10 min, remove the supernatant, resuspend the NK cell pellet with FBS freezing solution containing 10% DMSO, divide the NK cell suspension into target doses, quickly place in a -80 °C refrigerator for 24 h, and then transfer to liquid nitrogen for cryopreservation for later use.
[0037] Example 7 NK cell concentration, purity and proliferation test detection 7.1. NK cell purity detection Taking the NK cells cultured in Examples 4 and 6 as an example, samples were taken on the 0th day, the 3rd day, the 5th day, the 7th day, the 9th day, and the 11th day of culture for flow cytometry detection.
[0038] For each test, the total number of cells in the test sample of each embodiment was 5×10 5 , washed twice with PBS; added flow cytometry antibodies FITC-labeled mouse anti-human CD3 antibody and PE / Cyanine7-labeled mouse anti-human CD56 antibody (both products of BioLegend) respectively; incubated at room temperature for 20 minutes, washed twice with PBS, and the cells were analyzed by flow cytometry. The purity ratio of NK cells obtained by PBMC culture was tested. The test results are shown in Table 1 and Figure 2 As shown; among them, in the PBMC cells used in Example 4 and Example 6, the initial NK ratio was only about 8%.
[0039] Table 1 NK cell concentration ratio statistics
[0040] From Table 1 and Figure 2 It can be seen that the purity of the NK cells prepared in Example 4 is 99%, which is better than the purity of the NK cells prepared in Example 6 (85%); this is because, compared with the trophoblast cells K562 in Example 6, the trophoblast cells HEK293 used in Example 4 can better activate the rapid proliferation and growth of NK cells.
[0041] 7.2. NK cell purity detection The NK cells obtained by activating the PBMC in Example 4 through the trophoblast HEK293 cells were sampled on the 11th day for flow cytometry detection of the CD3-CD56+ ratio. The test results are as follows: Figure 3 As shown; The NK cells obtained by activating the PBMC in Example 6 through the trophoblast K652 culture were sampled on the 11th day for flow cytometry detection of the CD3-CD56+ ratio. The test results are as follows Figure 4 shown.
[0042] Depend on Figure 3 and 4 It can be seen that Figure 3 The CD3-CD56+ ratio of NK cells was 99.44%. Figure 4 The CD3-CD56+ ratio of NK cells was 85.07%; the test results showed that a large number of NK cells without CD3+ and with high purity were obtained in Example 4, while in Example 6, T cells with a CD3+CD56- ratio of 10.64% were also contained; that is, compared with the trophoblast cells K652, the trophoblast cells HEK293 cells can better activate the rapid proliferation and growth of NK cells.
[0043] 7.2. NK cell growth detection Taking the NK cells cultured in Examples 3 to 6 as an example, samples were taken and counted on day 0, day 3, day 5, day 7, day 9, and day 11 during the culture process. The NK cell counting results are shown in Tables 2 and Figure 5 In the PBMC cells used in Examples 4 and 6, the initial NK ratio was only about 8%; therefore, the number of NK cells on day 0 was 2.5×10 6 ×8%=0.2×10 6 Among them, Figure 5 It is drawn from the data in Table 2.
[0044] Table 2 NK cell count statistics
[0045] Accordingly, the results of the expansion multiples of NK cells during in vitro culture are shown in Tables 3 and Figure 6 As shown; among them, Figure 6 It is drawn from the data in Table 3 Table 3 NK cell expansion multiple statistics
[0046] From Tables 2, 3 and Figure 5 , 6 It can be seen that: 1) The growth (or expansion multiple) of NK cells prepared in Examples 3 and 4 is better than that of NK cells prepared in Examples 5 and 6; this is because irradiated mIL-21 and 4-1BBL gene-modified HEK293 cells as trophoblast cells can better activate the rapid expansion and growth of NK cells; 2) When the genetically modified HEK293 cells treated with the same irradiation were used as trophoblast cells, the growth (or expansion multiple) of the NK cells prepared in Example 4 was superior to the growth (or expansion multiple) of the NK cells prepared in Example 3.
[0047] In summary, trophoblast cells derived from irradiated gene-modified HEK293 cells are superior to trophoblast cells derived from K562 cells in terms of the in vitro expansion ability of NK cells and the purity of expanded NK cells.
[0048] Example 8 Tumor Cytotoxicity of NK Cells After the 11th day of cell culture in Example 4 and Example 6 (Comparative Example 2), NK cells (effector cells) were sampled and incubated with 1×10 4 K562 cells (target cells) were co-cultured in a 96-well plate, and the ratio of effector cells to target cells (E:T) was 10:1, 5:1, 1:1, 0.5:1, and 0.1:1. Each experiment was repeated 3 times. After 4 hours of co-culture, 100 μL / well of luciferase substrate was added to the 96-well plate, the mixed cells were resuspended and mixed, and the RLU (relative light unit) was immediately measured by a multifunctional microplate reader for 1 second. The luciferase quantitative killing efficiency evaluation method was used to evaluate the killing effect of NK cells on K562 in vitro. The killing ratio calculation formula is as follows: NK cell killing rate = 100% × (control well reading - experimental well reading) / control well reading; The calculation results are as follows Figure 7 As shown, under the same E:T, the killing ability of Example 4 is higher than that of Example 6, which indicates that the tumor killing ability of feeder layer cells derived from HEK293 cells is better than that of feeder layer cells derived from K562 cells.
[0049] In summary, the HEK293 cells overexpressing mIL-21 and 4-1BBL genes constructed in the present invention are stably transfected cells, which significantly improve the in vitro expansion ability of NK cells, the purity of amplified NK cells, and the tumor cell killing ability.
[0050] It should be understood that the above description of the preferred embodiments of the present invention is relatively detailed and cannot be regarded as limiting the scope of patent protection of the present invention. The scope of patent protection of the present invention shall be based on the attached claims.
Claims
1. A genetically modified HEK293 cell, characterized in that: The genes encoding mIL-21 and 4-1BBL were inserted into the genes of the cells.
2. The HEK293 cell according to claim 1, characterized in that The amino acid sequences corresponding to the coding genes of mIL-21 and 4-1BBL are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.
3. A method for preparing HEK293 cells as claimed in claim 1 or 2, characterized in that: The steps include: Construction of lentiviral expression plasmid: constructing the coding gene fragments of mIL-21 and 4-1BBL on lentiviral plasmids respectively to obtain lentiviral expression plasmids; Lentivirus packaging: adding lentivirus envelope plasmid pMD2.G, lentivirus expression plasmid, auxiliary plasmid pMDLg / pRRE, auxiliary plasmid pRSV-Rev and transfection reagent to 293T cell solution for lentivirus packaging, collecting supernatant, filtering and concentrating to obtain lentivirus concentrate; Cultivation of gene-modified HEK293 cells: HEK293 cells were inoculated into the wells of a culture plate containing a culture medium, the lentiviral concentrate was added into the wells of the culture plate, and the cells were incubated in an incubator at 37° C. and 5% CO 2 ; after cessation of the culture, HEK293 cells modified with the mIL-21 and 4-1BBL genes were obtained.
4. The method for preparing HEK293 cells according to claim 3, characterized in that: The following steps are also included: Irradiation treatment of gene-modified HEK293 cells: The mIL-21 and 4-1BBL gene-modified HEK293 cells were irradiated with 100 Gy of γ-rays for 0.5 h to obtain irradiated HEK293 cells; Transfer to liquid nitrogen for storage.
5. The method for preparing HEK293 cells according to claim 3 or 4, characterized in that: The lentivirus packaging step further includes the following steps: The lentiviral envelope plasmid pMD2.G, the lentiviral expression plasmid, the auxiliary plasmid pMDLg / pRRE, and the auxiliary plasmid pRSV-Rev were mixed to obtain a four-plasmid mixture; wherein the mass ratio of the lentiviral envelope plasmid pMD2.G, the lentiviral expression plasmid, the auxiliary plasmid pMDLg / pRRE, and the auxiliary plasmid pRSV-Rev was 1:4:3:2; According to a volume ratio of 1:2, Lipofectamine 3000 transfection agent was added to the four-plasmid mixture and mixed evenly, and the mixed four plasmids were transferred into 293T cells to obtain a lentiviral vector solution overexpressing mIL-21 and 4-1BBL; The lentiviral vector solution is centrifuged, the supernatant is collected, filtered and concentrated to obtain a lentiviral concentrate.
6. The method for preparing HEK293 cells according to claim 3 or 4, characterized in that: The culture of the genetically modified HEK293 cells comprises the following steps: The cell density per well was 1×10 5 HEK293 cells were inoculated into the wells of the culture plate, and each well was filled with DMEM medium containing 10% FBS, and cultured in an incubator at 37°C and 5% CO2 for 24 hours; After 24 h of culture, the culture medium waste liquid was discarded, and 300 μL of fresh serum-free DMEM medium was added to the wells of the culture plate, and the culture was continued for 1 h; After 1 hour of incubation, according to MOI=5, the lentiviral concentrate was added to the wells of the culture plate and culture was continued for 6 hours; After the 6-hour incubation, 700 μL of DMEM medium containing 10% FBS was added to the wells of the culture plate and the culture was continued for 72 hours; After 72 hours of culture, 1 ml of DMEM medium containing 1 ug / mL puromycin and 10% FBS was added to the wells of the culture plate, and culture was continued for 24 hours. After the culture was completed, HEK293 cells modified with mIL-21 and 4-1BBL genes were obtained.
7. A method for culturing NK cells, characterized in that: The steps include: On day 0, take 2.5×10 6 PBMC or NK cells isolated from peripheral blood and 5×10 6 The HEK293 cells irradiated as claimed in claim 4 are added into a culture bottle containing NK cell culture medium, and statically cultured in an incubator at 37° C. and 5% CO 2 ; From the 3rd day to the 11th day, fresh NK cell culture medium is added to the culture flask every day; on the 7th day, HEK293 cells irradiated as described in claim 4 need to be added to the culture flask until the end of the 11th day of culture, and the cultured NK cells are collected.
8. The NK cell culture method according to claim 7, characterized in that: After obtaining NK cells on day 11, the following treatments are also included: The collected NK cells were transferred to a centrifuge tube, centrifuged at 700 g for 10 min, and the supernatant was removed; the NK cell pellet was resuspended with a freezing solution, and the obtained NK cell suspension was dispensed into freezing tubes, transferred to liquid nitrogen, and stored for later use.
9. The NK cell culture method according to claim 7, characterized in that: From the 3rd day to the 11th day, fresh NK cell culture medium was added every day to maintain the cell density at 1.0×10 6 On the 7th day, the number of HEK293 cells treated with the irradiation was 3×10 7 indivual.
10. The NK cell culture method according to any one of claims 7 to 9, characterized in that: The NK cell culture medium is 581 culture medium, which contains heat-inactivated human AB serum with a final concentration of 5 v / v% and IL-2 with a final concentration of 200 U / mL.